In the field of modern poultry farming, prevention of diseases by vaccination is a major means for sanitation regardless of a kind of chicks, i.e. a chick for breeding, a chick for laying eggs or a chick for meat. The vaccination, however, has to be done so frequently that personnel expenses become much higher to cause an economical disadvantage for a poultry farmer. In order to avoid this disadvantage, one can contemplate to simply mix several known vaccines. However, there is a problem that an interference occurs between viruses in case of a mixture of live vaccines and there is also a limitation in mixing amount in case of a mixture of inactivated vaccines. In addition, in case of a mixture of a live vaccine and an inactivated vaccine, there is observed a titer decrease due to an adsorption of a live vaccine antigen to a gel (adjuvant).
Recently, taking into account the above situations, alternative method has been attempted to employ a virus vector, i.e. multiple genes of vaccine antigens are incorporated into a single virus to prepare a multivalent live vaccine. This method makes it possible to prepare a multivalent live vaccine without inducing the interference between viruses or the increase of inoculation amount in case of the mixture of inactivated vaccines as mentioned above.
Hitherto, a research on the use of a virus as a vector has already been conducted in the preparation of vaccines for various viruses such as vaccinia virus, adenovirus, herpes simplex virus, retrovirus, and the like, and Hepatitis B surface antigen (HBs antigen) or glyco-proteins of rabies virus or varicella zoster virus have successfully been expressed in vitro. However, some of these viruses (other than vaccinia virus) are a virus having an oncogencity and hence the administration of these viruses to human or animals is restricted and not practical in viewpoint of safety. In addition, even if the virus itself is safe, it cannot be used effectively as a virus vector for birds, at which the present invention is aimed, since the birds to be inoculated are not an original host of the virus.
Besides, use of avian poxvirus (e.g. chick fowlpox virus) as a vector has been suggested and the virus has already been studied for use as a virus vector. It is reported that an exogenous gene can be incorporated into the virus DNA [Saeki et al., Abstract of the 35th Meeting of Japan Virology Society, page 209 (1987)]. However, in the modern poultry field, immune against fowlpox lasts for only short period of time, and hence, several inoculations of a vaccine virus (attenuated fowlpox virus or pigeon pox virus) are usually required during the breeding of chick. Consequently, when the poxvirus is used as the virus vector, a frequent vaccination is still required even though a virus vector wherein plural antigens are incorporated is prepared and used as a vaccine. In addition, in case of a poxvirus vector, it is known that the growth of poxvirus itself is greatly inhibited by a maternal antibody against the poxvirus and hence a sufficient immune response against the inserted antigens cannot be obtained.
Marek's disease is a malignant tumor whose outbreak can be prevented only by vaccination. The prevention mechanism is considered that when the host birds such as chick is permanently infected with the vaccine virus, humoral and cell-mediated immunities against Marek's disease virus are induced and maintained through life of the host, and thereby tumorigenesis by virulent virus is suppressed. This virus vaccine is usually administered in the form of live cells infected with the virus and characterized by that it can be administered to a new-born chick since the virus propagates via the cell-to-cell infection and hardly affected by the maternal antibody.
In consideration of the above-mentioned characteristics of the Marek's disease virus, in recent years, a multivalent vaccine has been developed using the Marek's disease virus as a vector. In order to prepare the multivalent live vaccine in which Marek's disease virus, the virus having much more excellent properties than those of other virus vectors, is utilized as a vector, it is necessary to find out the site suitable for incorporation of an exogenous gene or the removable region on the Marek's disease virus DNA.
Hitherto, the thymidine kinase (TK) gene and the gA gene on the Marek's disease virus DNA have been studies as the site for incorporation of an exogenous gene. However, it has been reported that the loss of the thymidine kinase activity due to mutation in the TK gene reduces a viral growth [P. Bandyopadyay et al. (1987), 12th INTERNATIONAL HERPESVIRUS WORKSHOP] and a recombinant virus has not been reported wherein an exogenous gene is incorporated into the TK gene. As to the gA gene, it has been reported that a recombinant virus wherein the LacZ gene is incorporated into the gA gene is unstable and cannot be purified [Kato Atsushi et al. (1991), 111th meeting of the Japan Veterinary Society]. Therefore, both TK and gA genes are not practical.
The gA as well as gB are one of major glycoproteins produced by the virus. Although it is known that inoculation of gB induces the production of a neutralizing antibody in animal body, it has not yet been observed by inoculation of gA, nevertheless, it is expected that gA causes a cellular immunization. Therefore, if the Marek's disease virus is desired to have both functions as a vector and as a vaccine, the insertion of an exogenous gene into this gA gene to mutate the gA gene is undesirable since this will deteriorate the function as a vaccine.
Under the circumstances, the present inventors had intensively studied as to less analyzed gene in order to prepare an effective recombinant Marek's disease virus, and as a result, have already found that the recombinant Marek's disease virus could be obtained by using a BamHI--H fragment of the Marek's disease virus type I gene (the 8th fragment from the biggest prepared by digesting the Marek's disease virus gene with the restriction enzyme BamHI) (EP 361182A).